Tannase-mediated biotransformation assisted separation and purification of theaflavin and epigallocatechin by high speed counter current chromatography and preparative high performance liquid chromatography: A comparative study

Abstract

A large scale isolation and purification of theaflavin (TF) and epigallocatechin (EGC) has been successfully developed by tannase-mediated biotransformation combining high-speed countercurrent chromatography. After tannase hydrolysis of a commercially available theaflavins extract (TE), the content of TF and EGC in tannase-mediated biotransformation product (TBP) achieved approximately 3 times enrichment. SEM studies revealed smooth tannase biotransformation and the possibility of recovery of the tannase. A single 1.5 hours' HSCCC separation for TF and EGC employing a two-phase solvent system could simultaneously produce 180.8 mg of 97.3% purity TF and 87.5 mg of 97.3% purity EGC. However, a preparative HPLC separation of maximum injection volume containing 120 mg TBP prepared 11.2 mg TF of 94.9% purity and 7.7 mg EGC of 89.9% purity. HSCCC separation demonstrated significant advantages over Prep HPLC in terms of sample loading size, separation time, environmental friendly solvent systems, and the production.

Innovative Approaches for Recovery of Phytoconstituents from Medicinal/Aromatic Plants and Biotechnological Production

“…A single run, stepwise elution with a two phase solvent system was composed of n-hexane-ethyl acetate-methanol-water at the volume ratios of 1:1:0.7:1 (v/v) and 1:1:1.2:1 (v/v) at 1.5 mL/min flow rate and 900 rpm revolution speed resulted in 56% retention of the stationary phase and yielded 4α,5α-dihydroxy norkurarinone, 7-methoxyl-4α,5α-dihydroxy norkurarinone, 6α-hydroxykurarinone, and norkurarinone with purities over 94%. Further, Xia et al [54] elaborated the efficient HSCCC separation procedure for theaflavins obtained in the tannase mediated product (TBP). The maximum sample loading enabled obtaining theaflavin, epigallocatechin, and epicatechin with purity over 96%.…”

Efficient enzymatic synthesis of theaflavin and its production mechanism

“…In addition to the parameters for TF synthesis, isolation and purification are also critical to determine the yield. Until now, various absorbents and purification techniques have been reported to isolate TFs monomers, such as polyamide, Diaion HP20SS, octadecylsilyl, Sephadex LH‐20, silica column chromatography, high‐speed counter–current chromatography, and preparative high‐performance liquid chromatography (P‐HPLC) (Ding et al., 2017; Kusano et al., 2015; Tanaka & Matsuo, 2020; Xia et al., 2016).…”